Asian Pacific Journal of Tropical Disease

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1 Asian Pac J Trop Dis 2014; 4(Suppl 1): S263-S267 S263 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Disease journal homepage: Document heading doi: /S (14) 襃 2014 by the Asian Pacific Journal of Tropical Disease. All rights reserved. High helmintic infection of the European grass snake, Natrix natrix and the dice snake, Natrix tessellate (Serpentes: Colubridae) from Iran Mohammad Reza Yossefi 1, Reza Nikzad 2, Mohammad Nikzad 2, Ali Mousapour 2, Shahab Ramazanpour 2, Mohammad Taghi Rahimi 2,3* 1 Department of Veterinary Parasitology, Islamic Azad University, Babol Branch, Babol, Iran 2 Young Researcher Club, Islamic Azad University, Babol Branch, Babol, Iran 3 Student Research Committee, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran PEER REVIEW Peer reviewer Mahdi Darvishi, Assistant Professor, Department of Pathology, Veterinary Faculty, Semnan, Iran. Tel: Fax: darvishimahdi2000@gmail.com Comments In general, this is a good study in which authors try to determine the helminthic parasites of two widely distributed snakes including N. natrix and N. tessellate in north of Iran. The results are valuable and important. The survey was conducted for the first time on N. tessellate in Iran and this is new host and locality records of parasitic helminthes including T. assula, O. europaea and R. fuscovenosa in the country. Details on Page S266 ABSTRACT Objective: To determine the helminth parasites of Natrix natrix Linnaeus, 1758 (N. natrix) and Natrix tessellata Laurenti, 1768 (N. tessellate) in north of Iran. Methods: Eighteen snakes including nine N. natrix and nine N. tessellata from Mazandaran Province, north of Iran were collected and examined during March 2011 to October 2011 for helminth parasites. The collected specimens were fixed and preserved in 70% ethanol. Results: All of the examined snakes (100%) were infected with parasitic helminth. The list of extracted helminths both in N. natrix and N. tessellata includes one Nematode: Rhabdias fuscovenosa (larva), one Digenea: Telorchis assula and one Cestoda: Ophiotaenia europaea. The infection rate of Ophiotaenia europaea, Telorchis assula and Rhabdias fuscovenosa (larva) from collected snakes were 100%, 83.3% and 61.1%, respectively. Moreover, in the current investigation the morphological characteristics of the collected helminths were described elaborately. Conclusions: This is the first survey on helminth parasites from N. tessellata in Iran and the helminthes are reported for the first time from this host in Iran. KEYWORDS Snake, Reptile, Helminth, Parasite, Iran 1. Introduction The grass snake, European grass snake, Natrix natrix Linnaeus, 1758 (Serpentes: Colubridae) (N. natrix), sometimes called the ringed snake or water snake occurs from Europe, Northwestern Africa and East to Middle Asia[1]. Grass snake is large reptile, reaching up to 190 cm in total length, though such large specimens are rare. *Corresponding author: Mohammad Taghi Rahimi, Student Research Committee, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Young Researchers Club, Islamic Azad University, Babol Branch, Babol, Iran. Tel: rahimimt@gmail.com Foundation Project: Supported by the Young Researchers Club, Islamic Azad University, Babol Branch, Iran (Grant No. BPJ1390D). Females are considerably larger than males, typically reaching a size of cm when fully grown. Males are approximately 20 cm shorter and significantly smaller in girth. Weight is about 240 g[2,3]. Natrix tessellate Laurenti, 1768 (Serpentes: Colubridae) (N. tessellate), the dice snake is distributed from Middle and Southern Europe to Western China[4]. Maximum size of this snake is cm. N. natrix and N. tessellate are semi-aquatic snakes Article history: Received 20 Dec 2013 Received in revised form 23 Dec, 2nd revised form 26 Dec, 3rd revised form 31 Dec 2013 Accepted 15 Jan 2014 Available online 28 Jan 2014

2 S264 and observed in most aquatic habitats in Iran. N. natrix and N. tessellate are the most common snakes in the north of Iran[5,6]. According to our literature review, despite of wide distribution of N. natrix and N. tessellata in Iran there is not comprehensive and adequate published data about parasitic helminthes of these snakes in Iran. Therefore, the current study was conducted to prepare a list of parasitic helminths of N. natrix and N. tessellata in Iran. East Ardabil Azarbaijan West Azarbaijan Kurdestan Zanjan Gilan Qazvin Hamedan TEHRAN Kermanshah Markazi Qom Lurestan llam Esfahan Khuzestan Caspian Sea Chahar Mahal va Bakhtiari Golestan Semnan Yazd North Razavi South 2. Materials and methods 2.1. Sampling Kohkiuyehva Boyerahmad Fars Bushehr Persian Gulf Kerman Hormozgan Sistan va Baluchestan A total of 18 snakes including nine (3 males, 6 females) N. natrix and nine (4 males, 5 females) N. tessellata were captured by hand from rural areas of the Mazandaran province, north of Iran, from March 2011 to October For the purpose of slaughtering an overdose of sodium pentobarbital (Nembutal ) were utilized. To look for endoparasites, dissection was performed from throat to vent meticulously. The body cavity was opened and the digestive tract was removed. After collecting the visible helminthes of each digestive tract (stomach, small intestine and large intestine), the contents were rinsed into sieve Mesh 70 to collect the remaining helminths. In addition, the following organs: esophagus, lungs, liver and urinary bladder separately were opened and examined for helminths at necropsy under a dissecting microscope. The extracted Digenea and Cestoda helminths were fixed and preserved in 70% ethanol, stained with carminic acid, dehydrated, then cleared and mounted in Canadabalsam. Nematodes were killed in hot saline solution and kept in a solution composed 70% ethanol and 5% glycerin. For examination they were cleared by lactophenol[7]. Identifications of helminths were performed according to available systematic keys[8-10] Study area Mazandaran province ( N, E) is located at the northern part of Iran and on the southern coast of the Caspian Sea (Figure 1). It covers an area of km 2 and its population is composed by inhabitants. This province has a particular geographical condition with moderate and subtropical climate with 70%-100% relative humidity, C average temperature and mm annual rainfall. This province is geographically divided into the coastal plains and the mountainous areas of Alborz Mountains Range. It has diverse ecosystems in many plains, prairies and forests[11]. Figure 1. Map of Iran, Western Asia. Green shows position of Mazandaran province. 3. Results Sea of Oman Both examined N. natrix and N. tessellata harbored 1 species of Digenea, Cestoda and Nematoda including Telorchis assula (T. assula), Ophiotaenia europaea (O. europaea) and Rhabdias fuscovenosa (R. fuscovenosa) (larva), respectively. All examined snakes (nine N. natrix and nine N. tessellata) showed infection by helmintic parasites. The Cestoda, O. europaea was observed in all examined snakes. 100% of female snakes were infected by at least two helminth parasites O. europaea and T. assula. The infection rate of O. europaea, T. assula and R. fuscovenosa (larva) from collected snakes were 100%, 83.3% and 61.1%, respectively. In addition, the mean weight of nine examined N. natrix and nine examined N. tessellate were recorded g and g. Table 1 depicts the extracted helminths from examined N. natrix and N. tessellate according to sex and organ involvement. The morphological characteristics of the extracted helminths were described elaborately below O. europaea The scolex was observed unarmed encompassing four suckers. Neck was observed short in length, strobile acraspedote; Immature proglottids had 390 µm length and µm width; mature proglottids was measured µm length and µm width; gravid proglottids µm long and µm wide; genital pore was distributed irregularly, opening in the middle of the proglottids; vagina anterior or posterior to the cirrus pouch; cirrus pouch was 461 µm length and 191µm width; testicle was observed in two separated parts; the ovary width was µm and vitellines

3 S265 Table 1 The extracted helminths from examined N. natrix and N. tessellate based on sex and infected organ (n=18). Helminth Site of infection N. natrix (%) scattered as a lateral line. Moreover, numerous lateral branches were seen distinctively in uterus of gravid proglottids (Figure 2) R. fuscovenosa N. tessellate (%) (3 males and 6 females) (4 males and 6 females) Male Female Male Female Total (%) T. assula (Trematoda) Small intestine 1/3 (33.3) 6/6 (100.0) 3/4 (75.0) 5/5 (100.0) 15/18 (83.3) O. europaea (Cestoda) Small intestine 3/3 (100.0) 6/6 (100.0) 4/4 (100.0) 5/5 (100.0) 18/18 (100.0) R. fuscovenosa (Larva) (Nematoda) Lung 3/3 (100.0) 3/6 (50.0) 1/4 (25.0) 4/5 (80.0) 11/18 (61.1) Oral opening was seen round and equipped with 6 weakly developed lips which had equal size and shape. Vulva was situated in equatorial position. In esophagus, a posterior bulb was observed. Female parasites were viviparous. Uteri was thin walled and filled with numerous eggs. The shell of egg was thin, smooth, clear and their egg size was and µm. Male parasites revealed a pair of almost equal spicules. A long spike was observed in the end of tail in both sexes (Figure 4). Figure 2. Gravid proglottid of O. europaea from N. tessellate T. assula The body was elongated with parallel margins, which appeared to be lingual shape. Prepharynex were observed in all of specimens. Furthermore, oesophagus of T. assula appeared moderately long. In addition, caeca seemed slender and gut was looked a little thicker at the end of body. Testes were observed oval, tandem and also contiguous. Concerning cirrus sac, it appeared elongate, large, either straight or sinuous, mainly was appeared in posterior to ventral sucker and contained sacculate seminal vesicle, elongate ejaculatory duct lined with prostatic cells. Cirrus often was seen protruded. Genital pore was seen in median part. Uterus showed descending and ascending symmetrical that firmly packed transverse coils between ovary and anterior testis. In middle of body, vitellarium forms contiguous lateral fields of small follicules (Figure 3). Figure 3. T. assula from N. tessellate. Figure 4. Tail of R. fuscovenosa (male) from N. natrix intestine. 4. Discussion In the current survey the following helminthes were collected from N. natrix and N. testella: O. eropaea and T. assula from small intestine and R. fuscovenosa larva from lungs. In this work, O. europaea was the most dominant species (100.0%) which is followed by T. assula (83.3%) and R. fuscovenosa larva (61.1%). According to our review literature, although there is not any report on helminth parasites of N. testella, two published studies were carried out on parasitic infection of N. natrix in Iran. In one case report described O. europaea from one N. natrix from north of Iran[5]. And also three helminths including O. eropaea, T. assula and R. fuscovenosa were found from three European grass snakes (N. natrix) in Iran[12]. A similar investigation was carried out on 21

4 S266 N. natrix at 6 locations in neighbor country, Turkey and the following helminthes were reported: Astiotrema monticelli, T. assula, Encyclometra colubrimurorum, Spirometra erinaceieuropaei and O. europaea from small intestine, Paralepoderma cloacicola from large intestine, R. fuscovenosa, Macrodera longicollis from lungs and Eustrongylides excisus (larva) from musculature. The prevalence rate of O. europaea from N. natrix and N. testella were recorded 81% and 85%, respectively that our results are in agreement with the above mentioned study[13]. These results indicate that O. europaea is the most prevailing parasite of examined snakes. Nematodes of the genus Rhabdias are considered as a common lung parasites of amphibians and reptiles with over 40 species described throughout the world[14]. R. fuscovenosa has been reported frequently from Palearctic and Nearctic snakes[9,14]. Histologic examination of N. natrix which was infected with R. fuscovenosa revealed vacuolar degeneration of the respiratory epithelium, hemorrhage, faveolae necrosis and obstruction[15]. The infection rate with R. fuscovenosa in our specimens was 61.1% which mostly corresponds with Yildrimhan results (43%)[13]. In addition, the Trematoda, Macrodera longicollis and the nematodes Hedruris sp. and Oxysomatium brevicaudatum were extracted from N. natrix in Turkey[16,17]. In another survey Renifer aniarum (Digenea: Reniferidae) was obtained from N. natrix in Calabria, southern Italy[18]. Severe granulomatous lesions in N. tessellate was attributed to Eustrongylides larvae and we did not observed this larva[19]. Members of the genus Telorchis are plagiorchiform intestinal parasites occurring worldwide. They are common inhabitant of North American freshwater turtles and occasionally, snakes and salamanders. T. assula were reported from Europe and Turkey[13,20]. From collected samples 83.3% harbored T. assula that is noticeably higher compared to Yildirimhan finding 52%. In this survey we observed the exactly same species in both examined N. natrix and N. tessellate. A similar diets and food preferences are considered as two causative agents of this helminthes coinfection of these snakes[1]. Based on the current investigation results, all snakes were shown infection by helminth parasites and this infection rate was considerably high in Mazandaran province. Moreover, a significant difference in the infection rate or parasite burden was generally noticed in accordance with the sex of the host. Female specimens harbored at least two helminth parasites O. europaea and T. assula. Authors draw a conclusion that this is the first comprehensive study on the helminth parasites of N. natrix and N. tessellate in order to prepare and complete the list of parasites fauna in Iran, but it represents the first report of O. europaea, T. assula and R. fuscovenosa from N. tessellate in Iran. Finally further helminthological investigations are highly suggested to provide enough information about reptiles and amphibians parasites in this area and other regions of country. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements We wish to thank to Veterinary Parasitology Museum in University of Tehran for their sincere cooperation. This work is supported by Young Researchers Club, Islamic Azad University, Babol Branch, Iran (Grant No. BPJ1390D). Comments Background Snakes are carnivorous reptiles of the suborder Serpentes. Snakes can carry bacteria, virus and parasites. Some of zoonotic parasitic pathogens cause diseases such as sparganosis, mesocestodiasis and pentastosomiasis. Among snakes N. natrix (European grass snake) Linnaeus, 1758 and N. tessellate (dice snake) Laurenti, 1768 have a considerable distribution in the world and particularly in Iran. N. natrix and N. tessellate are the two most common snakes in north of Iran due to having proper geographical condition of this area. Having enough parasitic information about these snakes and their potential risk of zoonotic disease is significant. Research frontiers The study was carried out on eighteen snakes encompassing nine European grass snakes and nine dice snakes that were collected from Mazandaran province, North Iran. The samples were dissected and all organs were scrutinized for helminth parasite. The obtained parasites were preserved, cleared, fixed and indentified. In addition, the morphological features of extracted helminthes were described elaborately. Related reports The findings of paper is in agreement with Halajian (2013) and Youssefi (2010) investigations on N. natrix in Iran. The infection rate of the present work is higher than the study which was carried out by Yildirimhan (2007) in Turkey. But nine helminthes species were recorded from N. natrix.

5 S267 Innovations & breakthroughs Innovation in the study is that although N. natrix and N. tessellate have a considerable distribution in Iran there is no information on N. tessellate helminthes parasite and there is just limited data on N. natrix in Iran. Therefore, authors tried to fill this gap which is so valuable point of view. Applications Due to lack of enough information about parasitic burden of N. natrix and N. tessellate the current study could fill this gap and also clarifies the condition of parasitic diseases in Iran. The work provides fundamental data on these reptiles which are necessary. Furthermore, the paper provides a comprehensive morphological description of parasites which help researchers for better identification. Peer review In general, this is a good study in which authors try to determine the helminthic parasites of two widely distributed snakes including N. natrix and N. tessellate in north of Iran. The results are valuable and important. The survey was conducted for the first time on N. tessellate in Iran and this is new host and locality records of parasitic helminthes including T. assula, O. europaea and R. fuscovenosa in the country. Authors gave a comprehensive morphological description of recovered helminthes which taxonomically are valuable. The findings indicate a high parasitic infection (100%) of examined snakes. And these results are so promising for further investigations for researchers in the future. References [1] Vlček P, Najbar B, Jablonski D. First records of the dice snake (Natrix tessellata) from the North-Eastern part of the Czech Republic and Poland. Herpetol Notes 2010; 3: [2] Reading CJ, Jofré GM. Habitat selection and range size of grass snakes Natrix natrix in an agricultural landscape in Southern England. Amphibia Reptilia 2009; 30(3): [3] Wisler C, Hofer U, Arlettaz R. Snakes and monocultures: habitat selection and movements of female grass snakes (Natrix natrix L.) in an agricultural landscape. J Herpetol 2008; 42(2): [4] Mebert K, editor. The dice snake, Natrix tessellata: biology, distribution and conservation of a Palaearctic species. Rheinbach, Germany: DGHT; [5] Youssefi MR, Hoseini SH, Mobedi I, Ahoo MB, Rajabloo M. Ophiotaenia europaea, from Natrix natrix of north of Iran. World J Zool 2010; 5: [6] Ahmadzadeh F, Mebert K, Ataei S, Rezazadeh E, Goli LA, Böhme W. Ecological and biological comparison of three populations of the dice snake (Natrix tessellata) from the Southern Caspian Sea Coast, Iran. Mertensiella 2011; 18: [7] Purwaningsih E. New host and locality records of snake intestinal nematode Kalicephalus spp in Indonesia. Asian Pac J Trop Biomed 2011; 1(2): [8] Gibbons LM. Keys to the nematode parasites of vertebrates: Supplementary volume. CABI Publishing, Wallingford, UK [9] Gibsons DI, Jons A, Bray RA. Keys to the Trematoda. CABI Publishing and Natural History Museum, London [10] Khalil LF, Jones A, Bray RA. Keys to the cestode parasites of vertebrates. Wallingford, UK: CABI Publishing; [11] Mahdavi SA, Raeesi A, Faraji L, Youssefi MR, Rahimi MT. A case of misdiagnose of malaria infection. Asian Pac J Trop Biomed 2013; 3(9): [12] Halajian A, Bursey CR, Goldberg SR, Gol SMA. Helminth parasites of the European glass lizard, Pseudopus apodus (Squamata: Anguidae), and European grass snake, Natrix natrix (Serpentes: Colubridae), from Iran. Comp Parasitol 2013; 80: [13] Yildirimhan HS, Bursey CR, Goldberg SR. Helminth parasites of the grass snake, Natrix natrix, and the dice snake, Natrix tessellata (Serpentes: Colubridae), from Turkey. Comp Parasitol 2007; 74: [14] Rakhshanpour A, Youssefi MR, Marhaba Z, Aryaiepour M, Mobedi I, Kia EB, et al. The morphological and morphometric study of Rhabditis axei from Atherurus macrourus (Asiatic Brush-Tailed Porcupine). Global Veterinaria 2012; 8(3): [15] Mihalca AD, Miclăuş V, Lefkaditis M. Pulmonary lesions caused by the nematode Rhabdias fuscovenosa in a grass snake, Natrix natrix. J Wildlife Dis 2010; 46: [16] Coil W, Kuntz RE. Records of trematodes collected in Turkey with the descriptions of new species in the families Lecithodendriidae and Plagiorchiidae. Proc Helminthol Soc Washington 1958; 25: [17] Schad GA, Kuntz RE, Wells WH. Nematode parasites from Turkish vertebrates: an annotated list. Canadian J Zool 1960; 38: [18] Santoro M, Tkach VV, Mattiucci S, Kinsella JM, Nascetti G. Renifer aniarum (Digenea: Reniferidae), an introduced North American parasite in grass snakes Natrix natrix in Calabria, southern Italy. Dis Aquat Organ 2011; 95: [19] Mihalca A, Fictum Škorič PM, Sloboda M, Kärvemo S, Ghira I, Carlsson M, et al. Severe granulomatous lesions in several organs from Eustrongylides larvae in a free-ranging dice Snake, Natrix tessellata. Vet Pathol 2007; 44: [20] Youssefi MR, Mousapour A, Nikzad R, Mobedi I, Rahimi MT. First report of Telorchis assula (Digenea: Telorchiidae) in three reptile species from north of Iran. World J Zool 2013; 8 (3):